Idiopathic Hemochromatosis (IH) is a heritable, recessive disorder linked to the HLA-A locus within the human Major Histocompatibility Complex (MHC) on chromosome 6p21.3. The disease is perhaps the most common genetic disorder carrier frequency of 0.10) whose hallmark is chronic and systemic iron accumulation. Our initial efforts have centered on the isolation of MHC polymorphisms and their utilization in positionally localizing the defect through genetic analysis. Based on linkage disequilibrium studies and the published description of a bona fide recombinant individual, it is now possible to unequivocally assign the IH gene to within a well-defined and approachable critical region of 250 kb extending from the HLA-H locus through HLA-F. This entire region is contained within overlapping cosmid and Yeast Artificial Chromosome clones (YACs) which have aided our laboratory in contributing to the creation of a physical map of this class I subregion. This proposal focuses on the cloning and characterization of candidate disease genes residing within the above genomic interval. using DNA probes rich in CpG dinucleotide sequences (indicative of genes) as well as two novel technologies utilizing whole YAC clones, we have already identified several multigene families and have cloned a truncated cDNA recognizing a 9.5kb transcript from the critical region which we believe to one of several strong IH gene candidates. During the funding period, each of the novel clones will be sequenced, aided in part by exon trapping experiments, and further characterized. Clones will serve as probes on Northern blots of intestinal mucosa and liver RNA derived from normals and affected individuals contained within our substantial patient population. In concert with Northern analyses, PCR probes will also be made based on cDNA sequences in order to test for mutations within pools of cDNA derived from patients and normals by either Single-Strand Conformation Polymorphism (SSCP) or Denaturing Gradient Gel Electrophoresis (DGGE) analyses. Results from this study should aid in the early detection and prevention of this common and debilitating disorder.